Introduction: Aging is associated with increased levels of circulating inflammatory markers and reduced muscle mass and strength. Objective: We investigated whether intake of protein-enriched milk for 12 weeks would influence markers of inflammation among adults ≥70 years of age with reduced physical strength. Methods: In a double-blind randomized controlled intervention study, subjects were randomly allocated into two groups, receiving a protein-enriched milk (2 × 20 g protein/d, n = 14, mean (±SD) age 76.9 ± 4.9 yrs) or an isocaloric carbohydrate drink (n = 17, age 77.7 ± 4.8 yrs) for 12 weeks. We measured serum and mRNA expression levels of inflammatory markers in PBMCs. Results: Significant differences in the mRNA expression of nuclear receptor subfamily, group H, member 3 (NR1H3, encoding the LXRα transcription factor) and interferon gamma (INFG) were observed between groups. The mRNA level of TNFRSF1A was significantly reduced, while the mRNA level of dipeptidyl-peptidase 4 (DPP4) was significantly increased, in the control group. The serum level of TNFα increased significantly in the control group, while sTNFRSF1A increased significantly in both groups, but with no significant differences between groups. Conclusion: Consumption of a low-fat, protein-enriched milk for 12 weeks had minor effects on inflammatory related markers in older adults compared to an isocaloric carbohydrate drink.

Background As an intracellular human pathogen, Mycobacterium tuberculosis (Mtb) is facing multiple stressful stimuli inside the macrophage and the granuloma. Understanding Mtb responses to stress is essential to identify new virulence factors and pathways that play a role in the survival of the tubercle bacillus. The main goal of this study was to map the regulatory networks of differentially expressed (DE) transcripts in Mtb upon various forms of genotoxic stress. We exposed Mtb cells to oxidative (H2O2 or paraquat), nitrosative (DETA/NO), or alkylation (MNNG) stress or mitomycin C, inducing double-strand breaks in the DNA. Total RNA was isolated from treated and untreated cells and subjected to high-throughput deep sequencing. The data generated was analysed to identify DE genes encoding mRNAs, non-coding RNAs (ncRNAs), and the genes potentially targeted by ncRNAs. Results The most significant transcriptomic alteration with more than 700 DE genes was seen under nitrosative stress. In addition to genes that belong to the replication, recombination and repair (3R) group, mainly found under mitomycin C stress, we identified DE genes important for bacterial virulence and survival, such as genes of the type VII secretion system (T7SS) and the proline-glutamic acid/proline-proline-glutamic acid (PE/PPE) family. By predicting the structures of hypothetical proteins (HPs) encoded by DE genes, we found that some of these HPs might be involved in mycobacterial genome maintenance. We also applied a state-of-the-art method to predict potential target genes of the identified ncRNAs and found that some of these could regulate several genes that might be directly involved in the response to genotoxic stress. Conclusions Our study reflects the complexity of the response of Mtb in handling genotoxic stress. In addition to genes involved in genome maintenance, other potential key players, such as the members of the T7SS and PE/PPE gene family, were identified. This plethora of responses is detected not only at the level of DE genes encoding mRNAs but also at the level of ncRNAs and their potential targets.

In addition to generating movement, skeletal muscle may have a function as a secretory organ. The aim of the present study was to identify novel proteins with signaling capabilities secreted from skeletal muscle cells. IL-7 was detected in media conditioned by primary cultures of human myotubes differentiated from satellite cells, and concentrations increased with incubation time. By immunoblotting and real-time RT-PCR IL-7 expression was confirmed at both protein and mRNA levels. Furthermore, with immunofluorescence and specific antisera, multinucleated myotubes were found to coexpress IL-7 and myosin heavy chain. During differentiation of human myotubes from satellite cells, IL-7 expression increased at mRNA and protein levels. In contrast, mRNA expression of the IL-7 receptor was 80% lower in myotubes compared with satellite cells. Incubations with recombinant IL-7 under differentiation conditions caused ∼35% reduction in mRNA for the terminal myogenic markers myosin heavy chain 2 (MYH2) and myogenin (MYOG), suggesting that IL-7 may act on satellite cells to inhibit development of the muscle fiber phenotype. Alternative routes of cell development were investigated, and IL-7 increased migration of satellite cells by 40% after 48 h in a Transwell system, whereas cell proliferation remained unchanged. In vivo, real-time RT-PCR analysis of musculus vastus lateralis (n = 10) and musculus trapezius (n = 7) biopsies taken from male individuals undergoing a strength training program demonstrated that after 11 wk mean IL-7 mRNA increased by threefold (P = 0.01) and fourfold (P = 0.04), respectively. In conclusion, we have demonstrated that IL-7 is a novel myokine regulated both in vitro and in vivo, and it may play a role in the regulation of muscle cell development.

Complement activation can cause tissue damage in cerebral stroke by the release of biologically potent activation products and impaired function of regulatory proteins. We investigated the constitutive and hypoxia–reoxygenation-dependent expression of complement receptor 1 (CD35), membrane cofactor protein (CD46), decay-accelerating factor (CD55), protectin (CD59), and complement C3a and C5a receptors (C3aR and C5aR) on human NT2-N neurons. The effect of hypoxia–reoxygenation on C3d-deposition on neurons and endothelial cells was also investigated. NT2-N neurons were examined by cellular enzyme-linked immunosorbent assay and immunofluorescence microscopy. Endothelial cells were examined by flow cytometry. Three hours 1% or 0.1% hypoxia and 21 h reoxygenation with 50% AB-serum were used to investigate the effect of hypoxia–reoxygenation on regulators and C3d-deposition. NT2-N neurons expressed significant amounts of CD59 (Clone H19/Clone BRIC229: p = 0.000006/p = 0.000003), CD46 (p = 0.00006), CD55 (p = 0.003) and C3aR (p = 0.00003). CD35 and C5aR were not significantly expressed. There were no effects of hypoxia–reoxygenation on any of the regulators or receptors after 1% hypoxia and reoxygenation. However, CD55 (p = 0.02) was down-regulated after 0.1% hypoxia and subsequent reoxygenation with AB-serum. There were no difference observed in the C3d-deposition during hypoxia–reoxygenation in either neurons or endothelial cells. In conclusion, human NT2-N neurons constitutively express C3aR, CD46, CD55 and, in particular, CD59. The cells may respond to locally produced C3a and, at the same time, be well protected against complement attack. Although severe hypoxia–reoxygenation may down-regulate CD55 expression, it does not seem to influence C3d-deposition

Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are critical regulators of neuronal differentiation. The expression, levels and activities of PKA subunits were studied prior to and during differentiation of the human neuronal precursor cell line NTera 2 (NT2). Undifferentiated NT2 cells expressed mainly cytoplasmic PKA type I, consisting of the regulatory subunit RIalpha and the catalytic subunit Calpha. Low levels of PKA type II consisting of RIIalpha or RIIbeta associated with Calpha were also detected, mainly in the cytoplasm and in the Golgi-centrosomal area. During retinoic acid-induced differentiation, the RIalpha and RIIalpha expressions remained in the cytoplasm, while we observed a strong upregulation of RIIbeta, located to the whole cytoplasm including neurite extensions. This upregulation coincided with increased PKA-specific activity accompanied by a strong induction of a number of neuronal-specific Cbeta splice variants that together with RIIbeta form novel PKAII holoenzymes. Formation of novel PKAII holoenzymes may imply specific PKA features which may have consequences for the process of neuronal differentiation and nerve cell function

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta,Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1,1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.

We report a role for HA95, a nuclear protein with high homology to the nuclear A-kinase anchoring protein AKAP95, in the regulation of nuclear envelope-chromatin interactions. Biochemical and photobleaching data indicate that HA95 is tightly associated with chromatin and the nuclear matrix/lamina network in interphase, and bound to chromatin at mitosis. HA95 resides in a complex together with lamin B receptor (LBR), lamina-associated polypeptide (LAP)2 and emerin, integral proteins of the inner nuclear membrane. Cross-linking experiments, however, illustrate a tight association of HA95 with LBR and LAP2 only. Intra-nuclear blocking of HA95 with anti-HA95 antibodies abolishes nuclear breakdown in a mitotic HeLa cell extract. The antibodies inhibit nuclear membrane breakdown and chromatin condensation - the latter independently of nuclear membranes. However, lamina disassembly is not affected, as judged by immunological analyses of A/C- and B-type lamins. In contrast, immunoblocking of HA95 bound to condensed chromosomes does not impair chromatin decondensation, nuclear membrane reassembly or lamina reformation. Our results argue for a role for HA95 in anchoring nuclear membranes and lamins to chromatin in interphase, and in releasing membranes from chromatin at mitosis. The data also suggest that HA95 is not involved in initial binding of membranes to chromatin upon nuclear reassembly. We propose that HA95 is a central platform at the chromatin/nuclear matrix interface implicated in regulating nuclear envelope-chromatin interactions during the cell cycle.

LH and FSH regulate via cyclic adenosine 3'5' cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (PKA), steroid biosynthesis is Leydig and Sertoli cells, respectively. Cyclic AMP also regulates a number of different cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the PKA. The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement, has raised the question of how specificity is maintained in the cAMP/PKA system. Here we describe features of this signaling pathway that may contribute to explain how differential effects of cAMP may be contributed to features of the PKA signaling pathway.

Selectivity in the action of cAMP may be mediated by compartmentalized pools of cyclic AMP-dependent protein kinase (PKA). PKA type II is directed to different subcellular loci by interaction of the type II regulatory subunits (RIIalpha, RIIbeta) with A-kinase anchoring proteins. In order to separately investigate the subcellular localization of PKA type II isozymes, monospecific antibodies to human RIIalpha and RIIbeta subunits of PKA were developed. We demonstrate that centrosomes bind both RIIalpha and RIIbeta. Centrosomes were the preferred intracellular anchoring site for RIIbeta. However, centrosomal localization of RIIbeta was observed only in some highly differentiated cells such as keratinocytes, granulosa cells, and macrophages and in all neoplastic cell lines examined. Centrosomal localization of RIIbeta was not observed in normal undifferentiated cells such as fibroblasts, myoblasts, and T and B cells. In contrast, RIIalpha was abundant in the Golgi area and in the trans-Golgi network (TGN). Furthermore, although RIIalpha appeared to colocalize with microtubules in the Golgi/TGN, extractions with nonionic detergent demonstrated that RIIalpha was mainly membrane-associated. In addition, alterations of microtubule dynamics with Nocodazole or Taxol affected the distribution of the detergent-extractable pool of RIIalpha, indicating that RIIalpha may localize with microtubule-associated vesicles. Thus, RIIalpha and RIIbeta clearly localize differently in the Golgi-centrosomal region. This indicates specific roles for PKA isozymes containing either RIIalpha or RIIbeta.

A large number of hormones, neurotransmitters, and other signaling substances that bind to G-protein coupled cell-surface receptors, converge their signals through one sole second messenger, 3', 5'-cyclic adenosine monophosphate (cAMP). Cyclic AMP has been shown to regulate cellular processes such as cell growth and differentiation, ion channel conductivity, synaptic release of neurotransmitters, and gene transcription. The principle intracellular target for cAMP in mammalian cells is the cAMP-dependent protein kinase (PKA). The fact that this broad specificity protein kinase mediates a number of discrete physiological responses following cAMP engagement has raised the question of how specificity is maintained in the cAMP/PKA system. It has been speculated to what extent specificity in cAMP signaling may be obtained by differential stimulation of multiple PKA holoenzymes that are targeted to various sites within a single cell through specific anchoring proteins.

A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A-kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21-22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled-coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII-binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA-buffer extracts of HeLa cells demonstrated co-precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co-purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene.